Ldlr variants and their use in compositions for reducing cholesterol levels

ABSTRACT

A recombinant vector having an expression cassette comprising a modified human low density lipoprotein receptor (hLDLR) gene is provided, wherein said hLDLR gene encodes a modified hLDLR comprising (a) one or more of the following amino acid substitutions: L318H, N295D, H306D, V307D, N309A, D310N, L318H, and/or L318D; or (b) an amino acid substitution of any of (a) in combination with one or more of the following amino acid substitutions: K796, K809R and/or C818A. Also provided are pharmaceutical compositions containing this vector and uses therefor in lowering cholesterol and/or treating familial hypercholesterolemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/306,419,filed Oct. 24, 2016, which is a National Stage Entry under 35 U.S.C. 371of International Patent Application No. PCT/US2015/027572, filed Apr.24, 2015, which claims the benefit under 35 USC 119(e) of U.S.Provisional Patent Application No. 61/984,620, filed Apr. 25, 2014 andU.S. Provisional Patent Application No. 62/022,627, filed Jul. 9, 2014.These applications are incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported in part by a grant from the National Institutesof Health, Heart. Lung and Blood Institute, P01-HL059407-15. The USgovernment may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Familial Hypercholesterolemia (FH) is an autosomal co-dominant disordercharacterized by absence of the receptor for low-density lipoproteins(LDLR); a single chain glycoprotein containing 839 amino acids in itsmature form. Hussain M M, et al, Annu Rev Nutr. 1999; 19:141-172.Patients with one abnormal allele, heterozygous FH (heFH) have moderateelevations in plasma LDL and suffer from premature coronary arterydisease (CAD), whereas homozygous FH patients (hoFH) have high serumcholesterol (LDL-C>24 mmol/L) that often results in the early onset oflife-threatening cardio vascular disease (CVD). Marais A D, Clin BiochemRev. 2004; 25:49-68. Current treatment options to reduce excess serumcholesterol include LDL apheresis [McGowan M P. J Clin Lipidol. 2013;7:521-26] and treatment with cholesterol lowering drugs. Hovingh G K, etal, Eur Heart J. 2013; 34:962-971. Orthotopic liver transplantation canlead to long term correction [Raal F J, 2012; 223:262-268], although, itis associated with substantial treatment related morbidity andmortality.

Liver-directed gene therapy using adeno-associated viral vectors (AAV)has been demonstrated in preclinical models to stably correct severalmetabolic disorders and is currently being pursued in clinical trialsfor treatment of hemophilia A and B, ornithine transcarbamylasedeficiency (OTC) and alpha1-antitrypsin (A1AT) deficiency. Wang L, etal, Mol Genet Metab. 2012; 105:203-211; Brantly M L, et al, Proc NatlAcad Sci USA. 2009; 106:16363-16368; Nathwani A C, et al., N Engl J Med.2011; 365:2357-2365; Ward N J, et al, Blood. 2011; 117:798-807].Recently, the effectiveness of AAV mediated gene therapy in correctingserum cholesterol levels in humanized mouse models of FH has beendemonstrated. Kassim S H, et al, PLoS One. 2010; 5:e13424. In thesemice, systemic administration of AAV8 expressing human LDLR (AAV8.hLDLR)led to a lowering of cholesterol to normal levels by day 7 which wassustained for over a year and led to regression of pre-existingatherosclerosis. However, AAV8.LDLR transduction was dose dependent andstatistically significant correction was only achieved at a vector doseof 1.5×10¹¹ GC/kg or above. For clinical gene therapy, minimizing thevector dose is critical for many reasons, including vector injectionvolume, toxicity, immune response and manufacturing and cost of goodsconstraints.

Hepatic LDLR expression is modulated by multiple pathways within thecell: LDLR transcription is regulated by the sterol response elementbinding proteins (SREBPs), and HMGcoA reductase inhibitors (statins)activate SREBPs by inhibiting cholesterol synthesis within hepatocytes[Blumenthal R S, Am Heart J. 2000; 139:577-583].

A second pathway of LDLR regulation, involving pro-protein convertasesubtilisin kexin 9 (PCSK9), was discovered based on human geneticsgain-of-function mutations that caused high LDL-C levels [Abifadel M, etal., Nat Genet. 2003; 34:154-156] and loss-of-function mutations thatcaused low LDL-C levels [Cohen J, et al., Nat Genet. 2005; 37:161-1653].The loss of PCSK9 function was associated with an 88% reduction incardiovascular disease and has led to the development of a new class ofcholesterol lowering drugs based on the inhibition of PCSK9 [FitzgeraldK, et al, Lancet. 2014; 383:60-68; Giugliano R P, et al, Lancet. 2012;380:2007-2017]. Patients with FH have significantly higher plasma levelsof PCSK9 [Raal F, et al., J Am Heart Assoc. 2013; 2:e000028].

A third pathway of LDLR regulation was discovered by Zelcher et al,[Zelcher N, et al., Science. 2009; 325:100-104] who demonstrated thedegradation of LDLR by IDOL (inducible degrader of LDLR). An E3ubiquitin ligase, IDOL was induced following activation of liver Xreceptors (LXRs) and subsequently interacted with the cytoplasmic tailof LDLR in mediating receptor ubiquitination and degradation.Furthermore, screening of subjects with low LDL-C identifiedloss-of-function mutations in IDOL that prevented degradation of LDLR[Sorrentino V, et al., Eur Heart J. 2013; 34:1292-1297].

Compositions useful for effectively lowering cholesterol in subjects,particularly those having familial hypercholesterolemia, are needed.

SUMMARY OF THE INVENTION

Novel engineered human low density lipoprotein receptor (hLDLR) variantsare provided herein, which have increased efficacy as compared to priorart “wild-type” LDLR, due to PCSK9 and/or IDOL resistance. Theseengineered variants of hLDLR are suitably characterized by a reducedaffinity for PCSK9 and/or IDOL, an increased systemic half-life, and areuseful for lowering cholesterol as compared to the native hLDLR. Thesevariants can be delivered to subjects in need thereof via a number ofroutes, and particularly by expression in vivo mediated by a recombinantvector such as a recombinant adeno-associated virus (rAAV) vector.

In some embodiments, a synthetic or recombinant vector comprising amodified hLDLr gene is provided. In some embodiments, the modified hLDLRgene encodes a modified hLDLR that reduces cholesterol followingexpression. In some embodiments, the modified hLDLR comprises one ormore amino acid substitutions that interfere with the wild-type hLDLRIDOL pathway and/or one or more amino acid substitutions which areresistant to degradation of hLDLR by interfering with the PCSK9 pathway.

In certain embodiments, the synthetic or recombinant vector encodes amodified hLDLR that comprises an amino acid substitution at amino acidposition N295, H306, V307, N309, D310, L318, L796, K809 and/or C818.These amino acid positions are based on the numbering of SEQ ID NO:1(the LDLR without the signal peptide). In a specific embodiment, the oneor more amino acid substitutions are N295D, H306D, V307D, N309A, D310N,L318H, and/or L318D, which are examples of amino acid substitutions thatinterfere with the wild-type hLDLR IDOL pathway. In another specificembodiment, the one or more amino acid substitutions are L769R, K809Rand/or C818A, which are examples of amino acid substitutions which areresistant to degradation of hLDLR by interfering with the PCSK9 pathway.In another specific embodiment, the recombinant vector encodes amodified hLDLR that comprises one or more of amino acid substitutionsN295D, H306D, V307D, N309A, D310N, L318H, and/or L318D in combinationwith one or more of amino acid substitutions L7696R, K809R and/or C818A(numbering based on SEQ ID NO:1).

In some embodiments, the recombinant vectors provided herein have anexpression cassette comprising the modified hLDLR. In some embodiments,the expression cassette comprises a promoter which specifically directsexpression of the modified hLDLR in liver cells.

In some embodiments, the recombinant vector is a recombinantadeno-associated virus (rAAV) vector. In some embodiments, the rAAV hasa capsid selected from AAV8, rh64R1, AAV9, or rh10. In a particularembodiment, an rAAV vector is provided that has an expression cassettecomprising a modified hLDLR gene, wherein said hLDLR gene encodes amodified hLDLR comprising an L318D amino acid substitution. In aspecific embodiment, the modified hLDLR further comprises a K809R and/orC818A amino acid substitution. In a specific embodiment, the rAAV vectorcomprises an expression cassette comprising a promoter whichspecifically directs expression of the modified hLDLr in liver cells.

In certain embodiments, the hLDLR gene encodes a modified hLDLr havingthree substitutions: L318D/K809R/C818A (numbering based on SEQ ID NO:1). Other combinations of substitutions may be selected.

In some embodiments, a pharmaceutical composition comprising apharmaceutically acceptable carrier and a recombinant vector asdescribed herein is provided. Also provided are methods for reducingcirculating cholesterol levels by administering to a subject in needthereof a recombinant vector described herein that has an expressioncassette, wherein said expression cassette further comprises regulatorycontrol sequences which direct expression of modified hLDLr in thesubject.

In yet another embodiment, methods for increasing the circulatinghalf-life of a hLDLR are provided, comprising modifying the hLDLR at oneor more amino acid positions (position numbers based on SEQ ID NO: 1)selected from: N295, H306, V307, N309, D310, L318, L7696, K809 and/orC818. In a specific embodiment, the hLDLR is modified to comprise one ormore amino acid substitutions selected from: N295D, H306D, V307D, N309A,D310N, L318H, and/or L318D. In another specific embodiment, the hLDLR isfurther modified to comprise a K769R, K809R and/or C818A amino acidsubstitution.

The recombinant vectors described above can be used in a regimen fortreating familial hypercholesterolemia.

Other aspects and advantages of the invention will be readily apparentfrom the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B provide the results of in vitro evaluation of LDLRvariants that escape hPCSK9 regulation. Plasmids expressing wild typehLDLR or one of the LDLR variants were co-transfected along with hPCSK9into HEK293 cells. 24 hours (hr) after transfection, cells were pulsedwith BODIPY™-LDL [Molecular Probes] for 2 hr and then evaluated by flowcytometry for fluorescent LDL positive cells. FIG. 1A is a bar chartshowing the percentage of BODIPY™-LDL positive cells when co-transfectedwith hLDLR or hLDLR along with hPCSK9. The experiment was controlled bytransfecting cells with an irrelevant plasmid (Mock). FIG. 1B is a barchart showing the fold change in BODIPY-LDL positive cells in hLDLR plushPCSK9 co-transfected cells relative to hLDLR only transfected cells.FIG. 1C is a bar chart showing in vivo results in a mouse modelexpressing mLDLR. The results indicate some level of interaction betweenhPCSK9 and mLDLR.

FIGS. 2A-2C provide results from a study showing in vivo overexpressionof hPCSK9 leads to an increase in serum cholesterol in animals dosedwith wild type hLDLR. LDLR^(−/−), APOBEC-1^(−/−) double knock-out (DKO)mice (n=4/group) were administered intravenously with a dose of 5×10¹⁰GC of AAV8.TBG.hLDLR or AAV8.TBG.hLDLR along with 5×10¹⁰ GC ofAAV9.hPCSK9 vector. Serum from animals before and 30 days after vectoradministration was analyzed for total serum cholesterol and HDLcholesterol. Non-HDL cholesterol levels were determined by subtractingthe HDL component from total cholesterol. FIG. 2A is a bar chart showingthe percent change in day 30 non-HDL serum cholesterol relative tobaseline levels in animals that received hLDLR with or without hPCSK9.FIG. 2B is a line graph showing the time course of hPCSK9 expression inserum from mice that received hLDLR or hLDLR plus hPCSK9. hPCSK9expression was evaluated using a sandwich ELISA. The reported referenceaverage hPCSK9 levels in humans is also shown on the graph. FIG. 2C isan immunoblot of hLDLR expression in mice treated with hLDLR or hLDLRwith hPCSK9. Total liver lysates from two representative animals pergroup were electrophoresed on a 4-12% gradient SDS gel and probed with apolyclonal anti-hLDLR goat polyclonal antibody. Mouse tubulin expressionwas used as a loading control. All values are expressed as mean±SEM.***p<0.001.

FIGS. 3A and 3B illustrate that mice transduced with hLDLR-L318D areresistant to hPCSK9 mediated regulation. DKO mice (n=4/group) wereco-transduced with 5×10¹⁰ GC of AAV8.hLDLR or hLDLR-L318D along with5×10¹⁰ GC of AA9.hPCSK9. Serum from animals pre and 30 days post vectoradministration was evaluated for total cholesterol and HDL cholesterol.FIG. 3A is a bar chart showing the percent change in day 30 non-HDLserum cholesterol levels relative to pre-vector administration in DKOmice co-administered with hLDLR or hLDLR-L318D, along with hPCSK9. FIG.3B show total liver lysates from two animals per group which wereelectrophoresed on a 4-12% SDS PAGE gel and probed for hLDLR expression.Mouse tubulin expression was used as a loading control. All values areexpressed as mean±SEM. ***p<0.001. ns p>0.05.

FIGS. 4A-4D illustrate AAV8.hLDLR-K809R\C818A escapes in vivo hIDOLmediated regulation. HEK293 cells were transiently transfected withplasmids expressing either hLDLR or hLDLR-K809R\C818A along with hIDOL.24 hr later, cells were pulsed with BODIPY-LDL for 2 hr and thenevaluated for fluorescent LDL uptake using a flow cytometer. FIG. 4A isa bar chart showing the percent BODIPY™-LDL positive cells transfectedwith hLDLR or hLDLR-K809R\C818A along with hIDOL. FIG. 4B is a linegraph showing data from LDLR^(+/−), Apobec^(−/−), Tg-hApoB100 (LAHB)heterozygous FH (heFH) mice (n=4) which were systemically administeredwith 1×10¹¹ GC of AAV9hPCSK9 vector. Time course of non-HDL cholesterollevels following vector administration. FIG. 4C is a bar chart showinghomozygous FH (hoFH) DKO mice (n=4\group) systemically administered with3×10⁹GC AAV8.hLDLR, or AAV8.hLDLR-K809R\C818A, along with AAV9.hIDOL5×10¹⁰ GC. Serum from animals pre- and 30 days post vectoradministration was evaluated for total serum cholesterol. Percent changein serum non-HDL levels at 30 day relative to pre-administrationbaseline levels. All values are expressed as mean±SEM.***p<0.0001.*p<0.05. ns p>0.05. FIG. 4D provides the total cell lysatesof transfected cells (FIG. 4A) electrophoresed on a 4-12% SDS gel andprobed using anti-hLDLR antibody. The location of mature (M) andprocessed (P) forms of LDLR along with the tubulin loading control isshown.

FIGS. 5A-5B illustrate the AAV8.hLDLR-L318D\K809R\C818A variant encodingthree amino acid substitutions escapes both PCSK9 and IDOL mediatedregulation. FIG. 5A illustrates the results in a study in which DKO mice(n=4) were intravenously administered with 3×10⁹ GC of hLDLR orhLDLR-L318D\K809R\C818A. Additional groups of mice also received asimultaneous administration of AAV9.hIDOL. Total serum cholesterollevels were evaluated before and 30 days after vector administration.Percent decrease in non-HDL cholesterol relative to baseline. Totalliver lysates from 2 representative animals per group wereelectrophoresed on a SDS PAGE gel and probed using an anti-hLDLRantibody along with tubulin as a loading control. FIG. 5B illustratesthe results following coadministration of AAV8.hLDLR (5×10¹⁰ GC) orhLDLR-L318D\K809R\C818A along with AAV9.hPCSK9 (5×10¹⁰ GC). Percentdecrease in day 30 non-HDL cholesterol relative to baseline is shownalong with an immunoblot of hLDLR expression in livers. n***p<0.001.

FIG. 6 is a bar chart illustrating the hLDLR activity of variants thatescape PCSK9 regulation in DKO mice. DKO mice (n=4/group) were injectedwith 3×10¹⁰ GC of AAV8.TBG.hLDLR or AAV8 vectors expressing one of ninehPCSK9 escape variants. Serum from animals was analyzed before and 30days after vector administration and percent reduction in non-HDLcholesterol at day 30 day compared to baseline is shown along with SD.

FIG. 7 is a bar chart illustrating that AAV.hLDLR overcome hIDOLmediated inhibition when administered at higher dose. DKO mice(n=4/group) were administered with of 5×10¹⁰ GC of AAV8.TBG.hLDLR orAAV8.TBG.hLDLR-K809R\C818A. Additional groups of mice received hLDLRalong with of 5×10¹⁰ GC of AAV9.TBG.hIDOL. Percent non-HDL cholesterollevels on day 30 compared to baseline is shown along with SD.

FIG. 8 is a bar chart illustrating the hLDLR activity of variants thatescape PCSK9 regulation in a LDLR −/−, ApoBec −/−, double-knock out amouse model. DKO mice (n=5/group) were injected (tail vein) with 5×10¹⁰GC of AAV8.TBG.hLDLR or AAV8 vectors expressing one of nine hPCSK9escape variants along with 5×10¹⁰ GC of AAV9.TBG.hPCSK9 vectors. Serumfrom animals was analyzed before and 30 days after vectoradministration. Percent reduction in non-HDL cholesterol at day 30 daycompared to baseline is shown along with SD. Control mice received onlythe LDLR vector without co-administration of PCSK9 (bars to left in eachpair).

DETAILED DESCRIPTION OF THE INVENTION

The novel engineered human low density lipoprotein receptor (hLDLR)variants described herein are characterized by increased half-life andincreased efficacy in decreasing cholesterol levels as compared to thenative hLDLR due at least in part to their ability to substantiallyavoid degradation by pro-protein convertase subtilisin kexin 9 (PCSK9)and/or substantially avoid degradation by the inducible degrader of LDLR(IDOL).

Delivery of these variants to subjects in need thereof via a number ofroutes, and particularly by expression in vivo mediated by a recombinantvector such as a rAAV vector, are described. Also provided are methodsof using these variants in regimens for lowering cholesterol levels insubject in need thereof, treating familial hypercholesterolemia,treating atherosclerosis, decreasing the risk of premature coronaryartery disease and/or decreasing early onset of cardio vascular disease.Advantageously, compositions provided herein are useful forsimultaneously targeting multiple pathways in these treatments andregimens.

As used herein, the term familial hypercholesterolemia (FH) refers to agenetic disorder of lipid metabolism. Unless otherwise specified herein,both homozygous FH (hoFH) subjects and heterozygous FH (heFH) subjectsare encompassed within the term FH.

As used herein, the term “lowering cholesterol levels” may encompassdecreasing serum cholesterol levels and/or decreasing low-densitylipoprotein levels (e.g., in plasma). Treating atherosclerosis mayinclude decreasing number and/or volume of plaques and/or preventingfurther accumulation of atherosclerotic plaques.

The amino acid sequence of the mature “wild-type” hLDLR (isoform 1) isreproduced herein as SEQ ID NO: 1 for convenience and provides areference for the numbering of the amino acid variants provided herein.While the sequence numbering provided herein refers to the mature hLDLRprotein (a single chain glycoprotein of 839 amino acids), it will beunderstood that wild-type hLDLR leader sequence (amino acids 1-21 of SEQID NO:2) may be used or a heterologous leader sequence may be selectedfor use in the constructs described herein. Additionally, or optionally,one or more of the other hLDLR isoforms 2, 3, 4, 5 and 6, the sequencesof which are available, e.g., fromhttp://www.uniprot.org/uniprot/P01130, and the amino acid substitutionsdescribed herein may be incorporated into these isoforms (see also, SEQID NO: 3-7 where the sequences of these isoforms are reproduced forconvenience). In the following descriptions, substitutions may bewritten as (first amino acid identified by single letter code)-residueposition #-(second amino acid identified by single letter code) wherebythe first amino acid is the substituted amino acid and the second aminoacid is the substituting amino acid at the specified position withreference to isoform 1; however, by conventional alignment steps, thecorresponding amino acid residues identified herein with respect to thenumbering of isoform 1 can be located in the other isoforms or hLDLRproteins identified herein.

The term “amino acid substitution” and its synonyms described above areintended to encompass modification of an amino acid sequence byreplacement of an amino acid with another, substituting, amino acid. Thesubstitution may be a conservative substitution. It may also be anon-conservative substitution. The term conservative, in referring totwo amino acids, is intended to mean that the amino acids share a commonproperty recognized by one of skill in the art. For example, amino acidshaving hydrophobic nonacidic side chains, amino acids having hydrophobicacidic side chains, amino acids having hydrophilic nonacidic sidechains, amino acids having hydrophilic acidic side chains, and aminoacids having hydrophilic basic side chains. Common properties may alsobe amino acids having hydrophobic side chains, amino acids havingaliphatic hydrophobic side chains, amino acids having aromatichydrophobic side chains, amino acids with polar neutral side chains,amino acids with electrically charged side chains, amino acids withelectrically charged acidic side chains, and amino acids withelectrically charged basic side chains. Both naturally occurring andnon-naturally occurring amino acids are known in the art and may be usedas substituting amino acids in embodiments. Methods for replacing anamino acid are well known to the skilled in the art and include, but arenot limited to, mutations of the nucleotide sequence encoding the aminoacid sequence. Reference to “one or more” herein is intended toencompass the individual embodiments of, for example, 1, 2, 3, 4, 5, 6,or more.

As described herein, the hLDLR variants provided herein are engineeredto reduce the PCSK9 degradation characteristic of the wild-type LDLR. Inone embodiment, the variant is a human LDLR having an amino acidsubstation at position 318, in which the native leucine (Leu) ismodified. In one example, the L318 is modified to histidine (His, H).However, other substitutions (e.g., an L318D) may be made at thisposition. Alternatively or additionally, other hLDLR variants resistantto PCSK9 degradation may be selected from among those identified herein.These may include, e.g., substitutions of N295, H306, V307, N309, and/orD310 (position numbers based on SEQ ID NO:1). Methods of determiningresistance to PCSK9 degradation and/or determining increased circulatinghalf-life as compared to the wild-type hLDLR are known in the art, andat least one these assays is illustrated in the examples below.

Additionally, the PCSK9-resistant LDLR variants described herein may befurther engineered to include resistance to degradation by IDOL.Suitable substitutions for conferring this characteristic includesubstitutions at position K796 (abbreviated K6 in sequence listing),K809 and C818. The substitutions illustrated herein are K809R and C818A.However, other IDOL-resistant substitutions may be selected. Methods ofdetermining resistance to IDOL degradation and/or determining increasedcirculating half-life as compared to the wild-type hLDLR are known inthe art, and at least one these assays is illustrated in the examplesbelow.

Other modifications to the hLDLR isoform 1 amino acid sequence, whichincorporate one or more of the above variants, are encompassed withinthe invention. For example, the corresponding modification to the aminoacid sequence of any of isoforms 2 (SEQ ID NO:3), isoform 3 (SEQ ID NO:4), isoform 4 (SEQ ID NO: 5), isoform 5 (SEQ ID NO: 6), and isoform 7(SEQ ID NO: 7) may be utilized. These isoforms are reproduced in theSequence Listing herein. In another example, the hLDLR variant describedherein may be engineered to contain the hLDLR leader sequence.Alternatively, a heterologous leader sequence may be engineered to theN-terminus of the hLDLR variant. Alternatively, still other variations,which may include up to about 5% variation (about 95% identity to about99.9% identity to the variant sequence, or about 97% to about 98%identity) to the hLDLR variants provided herein (excluding the leadersequence) may be selected which retain one or more of the therapeuticfunctions of the hLDLR variants described herein, and which arecharacterized by PCSK9-resistance and/or IDOL-resistance.

In the examples section of this description, while a number ofconstructs did escape PCSK9 regulation in initial in vitro screening,the studies focused on the L318D amino acid substitution. Among thevariants provided herein, the L318D modification has been demonstratedto confer protection from PCSK9 both in vitro and in vivo. In theexamples provided herein, L318D (position number based on SEQ ID NO:1,illustrative construct with leader sequence in SEQ ID NO: 26) conferredprotection following hepatic expression in mice overexpressing PCSK9 andled to a significant decrease in serum cholesterol; whereas, wild-typeLDLR was less efficient and more readily degraded by PCSK9.

As illustrated in the examples below, the K809R/C818A hLDLR doublemutant (position numbers based on SEQ ID NO:1, illustrative constructwith leader sequence in SEQ ID NO: 36] conferred protection followinghepatic expression in mice expressing hIDOL and led to a significantdecrease in serum cholesterol; whereas, wild-type LDLR was lessefficient and more readily degraded by IDOL. These data thus establishthat the amino acid modifications in the LDLR can also overcome in vivoIDOL mediated suppression. Factors that lead to LDLR degradation areexpected to be higher in subjects lacking endogenous receptor expressiondue to lack of a substrate to remove the inhibitors. The usefulness ofLDLR variants in overcoming negative cellular regulatory pathways, knownto exist in FH subjects, is demonstrated herein. The findings presentedhere demonstrate for the first time the successful use of an AAV encoded‘gain-of-function’ transgene in reducing cholesterol in humanized mousemodels expressing high levels of inhibitory factors which is useful ingene therapy products for FH.

In addition to the hLDLR protein variants provided herein, nucleic acidsequences encoding these hLDLR protein variants are provided. The codingsequences for these variants may be generating using site-directedmutagenesis of the wild-type nucleic acid sequence. Alternatively oradditionally, web-based or commercially available computer programs, aswell as service based companies may be used to back translate the aminoacids sequences to nucleic acid coding sequences, including both RNAand/or cDNA. See, e.g., backtranseq by EMBOSS,http://www.ebi.ac.uk/Tools/st/; Gene Infinity(http://www.geneinfinity.org/sms-/sms backtranslation.html); ExPasy(http://www.expasy.org/tools/). In one embodiment, the RNA and/or cDNAcoding sequences are designed for optimal expression in human cells.

Codon-optimized coding regions can be designed by various differentmethods. This optimization may be performed using methods which areavailable on-line, published methods, or a company which provides codonoptimizing services. One codon optimizing method is described, e.g., inU.S. Patent Application No. 61/817,110, which is incorporated byreference herein. Briefly, the nucleic acid sequence encoding theproduct is modified with synonymous codon sequences. Suitably, theentire length of the open reading frame (ORF) for the product ismodified. However, in some embodiments, only a fragment of the ORF maybe altered. By using one of these methods, one can apply the frequenciesto any given polypeptide sequence, and produce a nucleic acid fragmentof a codon-optimized coding region which encodes the polypeptide.

The terms “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of nucleic acidsequences refers to the bases in the two sequences which are the samewhen aligned for correspondence. The length of sequence identitycomparison may be over the full-length of the genome, the full-length ofa gene coding sequence, or a fragment of at least about 500 to 5000nucleotides, or as desired. However, identity among smaller fragments,e.g. of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired. Multiple sequence alignmentprograms are also available for nucleic acid sequences. Examples of suchprograms include, “Clustal W”, “CAP Sequence Assembly”, “BLAST”, “MAP”,and “MEME”, which are accessible through Web Servers on the internet.Other sources for such programs are known to those of skill in the art.Alternatively, Vector NTI utilities are also used. There are also anumber of algorithms known in the art that can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta™, a program in GCG Version 6.1. Fasta™ providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta™ with its default parameters (a word size of 6 and the NOPAMfactor for the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference.

The terms “percent (%) identity”, “sequence identity”, “percent sequenceidentity”, or “percent identical” in the context of amino acid sequencesrefers to the residues in the two sequences which are the same whenaligned for correspondence. Percent identity may be readily determinedfor amino acid sequences over the full-length of a protein, polypeptide,about 32 amino acids, about 330 amino acids, or a peptide fragmentthereof or the corresponding nucleic acid sequence coding sequencers. Asuitable amino acid fragment may be at least about 8 amino acids inlength, and may be up to about 700 amino acids. Generally, whenreferring to “identity”, “homology”, or “similarity” between twodifferent sequences, “identity”, “homology” or “similarity” isdetermined in reference to “aligned” sequences. “Aligned” sequences or“alignments” refer to multiple nucleic acid sequences or protein (aminoacids) sequences, often containing corrections for missing or additionalbases or amino acids as compared to a reference sequence. Alignments areperformed using any of a variety of publicly or commercially availableMultiple Sequence Alignment Programs. Sequence alignment programs areavailable for amino acid sequences, e.g., the “Clustal X”, “MAP”,“PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs.Generally, any of these programs are used at default settings, althoughone of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program which provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensivecomparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

In one embodiment, the nucleic acid sequences encoding the hLDLRvariants (e.g., LDLR variant gene) described herein are engineered intoany suitable genetic element, e.g., naked DNA, phage, transposon,cosmid, RNA molecule (e.g., mRNA), episome, etc., which transfers thehLDLR sequences carried thereon to a host cell, e.g., for generatingnanoparticles carrying DNA or RNA, viral vectors in a packaging hostcell and/or for delivery to a host cells in subject. In one embodiment,the genetic element is a plasmid. The selected genetic element may bedelivered by any suitable method, including transfection,electroporation, liposome delivery, membrane fusion techniques, highvelocity DNA-coated pellets, viral infection and protoplast fusion. Themethods used to make such constructs are known to those with skill innucleic acid manipulation and include genetic engineering, recombinantengineering, and synthetic techniques. See, e.g., Green and Sambrook,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (2012).

As used herein, an “expression cassette” refers to a nucleic acidmolecule which comprises the hLDLR variant coding sequences, promoter,and may include other regulatory sequences therefor, which cassette maybe engineered into a genetic element and/or packaged into the capsid ofa viral vector (e.g., a viral particle). Typically, such an expressioncassette for generating a viral vector contains the hLDLR sequencesdescribed herein flanked by packaging signals of the viral genome andother expression control sequences such as those described herein.

The expression cassette typically contains a promoter sequence as partof the expression control sequences. The illustrative plasmid and vectordescribed herein uses the liver-specific promoter thyroxin bindingglobulin (TBG). Alternatively, other liver-specific promoters may beused [see, e.g., The Liver Specific Gene Promoter Database, Cold SpringHarbor, http://rulai.schl.edu/LSPD, alpha 1 anti-trypsin (A1AT); humanalbumin Miyatake et al., J. Virol., 71:5124 32 (1997), humAlb; andhepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002 9(1996)]. TTR minimal enhancer/promoter, alpha-antitrypsin promoter, LSP(845 nt)25(requires intron-less scAAV). Although less desired, otherpromoters, such as viral promoters, constitutive promoters, regulatablepromoters [see, e.g., WO 2011/126808 and WO 2013/04943], or a promoterresponsive to physiologic cues may be used may be utilized in thevectors described herein.

In addition to a promoter, an expression cassette and/or a vector maycontain other appropriate transcription initiation, termination,enhancer sequences, efficient RNA processing signals such as splicingand polyadenylation (polyA) signals; sequences that stabilizecytoplasmic mRNA; sequences that enhance translation efficiency (i.e.,Kozak consensus sequence); sequences that enhance protein stability; andwhen desired, sequences that enhance secretion of the encoded product.Examples of suitable polyA sequences include, e.g., SV40, bovine growthhormone (bGH), and TK polyA. Examples of suitable enhancers include,e.g., the alpha fetoprotein enhancer, the TTR minimal promoter/enhancer,LSP (TH-binding globulin promoter/alpha1-microglobulin/bikuninenhancer), amongst others.

These control sequences are “operably linked” to the hLDLR genesequences. As used herein, the term “operably linked” refers to bothexpression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

The expression cassette may be engineered onto a plasmid which is usedfor production of a viral vector. The minimal sequences required topackage the expression cassette into an AAV viral particle are the AAV5′ and 3′ ITRs, which may be of the same AAV origin as the capsid, orwhich of a different AAV origin (to produce an AAV pseudotype). In oneembodiment, the ITR sequences from AAV2, or the deleted version thereof(AITR), are used for convenience and to accelerate regulatory approval.However, ITRs from other AAV sources may be selected. Where the sourceof the ITRs is from AAV2 and the AAV capsid is from another AAV source,the resulting vector may be termed pseudotyped. Typically, an expressioncassette for an AAV vector comprises an AAV 5′ ITR, the hLDLR codingsequences and any regulatory sequences, and an AAV 3′ ITR. However,other configurations of these elements may be suitable. A shortenedversion of the 5′ ITR, termed AITR, has been described in which theD-sequence and terminal resolution site (trs) are deleted. In otherembodiments, the full-length AAV 5′ and 3′ ITRs are used.

The abbreviation “sc” refers to self-complementary. “Self-complementaryAAV” refers a plasmid or vector having an expression cassette in which acoding region carried by a recombinant AAV nucleic acid sequence hasbeen designed to form an intra-molecular double-stranded DNA template.Upon infection, rather than waiting for cell mediated synthesis of thesecond strand, the two complementary halves of scAAV will associate toform one double stranded DNA (dsDNA) unit that is ready for immediatereplication and transcription. See, e.g., D M McCarty et al,“Self-complementary recombinant adeno-associated virus (scAAV) vectorspromote efficient transduction independently of DNA synthesis”, GeneTherapy, (August 2001), Vol 8, Number 16, Pages 1248-1254.Self-complementary AAVs are described in, e.g., U.S. Pat. Nos.6,596,535; 7,125,717; and 7,456,683, each of which is incorporatedherein by reference in its entirety.

An adeno-associated virus (AAV) viral vector is an AAV DNase-resistantparticle having an AAV protein capsid into which is packaged nucleicacid sequences for delivery to target cells. An AAV capsid is composedof 60 capsid protein subunits, VP1, VP2, and VP3, that are arranged inan icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20,depending upon the selected AAV. AAV serotypes may be selected assources for capsids of AAV viral vectors (DNase resistant viralparticles) including, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2,AAV7, AAV8, AAV9, rh10, AAVrh64R1, AAVrh64R2, rh8 [See, e.g., USPublished Patent Application No. 2007-0036760-A1; US Published PatentApplication No. 2009-0197338-A1; EP 1310571]. See also, WO 2003/042397(AAV7 and other simian AAV), U.S. Pat. Nos. 7,790,449 and 7,282,199(AAV8), WO 2005/033321 and U.S. Pat. No. 7,906,111 (AAV9), and WO2006/110689], and rh10 [WO 2003/042397] or yet to be discovered, or arecombinant AAV based thereon, may be used as a source for the AAVcapsid. These documents also describe other AAV which may be selectedfor generating AAV and are incorporated by reference. In someembodiments, an AAV cap for use in the viral vector can be generated bymutagenesis (i.e., by insertions, deletions, or substitutions) of one ofthe aforementioned AAV Caps or its encoding nucleic acid. In someembodiments, the AAV capsid is chimeric, comprising domains from two orthree or four or more of the aforementioned AAV capsid proteins. In someembodiments, the AAV capsid is a mosaic of Vp1, Vp2, and Vp3 monomersfrom two or three different AAVs or recombinant AAVs. In someembodiments, an rAAV composition comprises more than one of theaforementioned Caps.

For packaging an expression cassette into virions, the ITRs are the onlyAAV components required in cis in the same construct as the gene. In oneembodiment, the coding sequences for the replication (rep) and/or capsid(cap) are removed from the AAV genome and supplied in trans or by apackaging cell line in order to generate the AAV vector. For example, asdescribed above, a pseudotyped AAV may contain ITRs from a source whichdiffers from the source of the AAV capsid. Additionally oralternatively, a chimeric AAV capsid may be utilized. Still other AAVcomponents may be selected. Sources of such AAV sequences are describedherein and may also be isolated or obtained from academic, commercial,or public sources (e.g., the American Type Culture Collection, Manassas,Va.). Alternatively, the AAV sequences may be obtained through syntheticor other suitable means by reference to published sequences such as areavailable in the literature or in databases such as, e.g., GenBank®,PubMed®, or the like.

Methods for generating and isolating AAV viral vectors suitable fordelivery to a subject are known in the art. See, e.g., U.S. Pat. Nos.7,790,449; 7,282,199; WO 2003/042397; WO 2005/033321, WO 2006/110689;and U.S. Pat. No. 7,588,772 B2]. In a one system, a producer cell lineis transiently transfected with a construct that encodes the transgeneflanked by ITRs and a construct(s) that encodes rep and cap. In a secondsystem, a packaging cell line that stably supplies rep and cap istransiently transfected with a construct encoding the transgene flankedby ITRs. In each of these systems, AAV virions are produced in responseto infection with helper adenovirus or herpesvirus, requiring theseparation of the rAAVs from contaminating virus. More recently, systemshave been developed that do not require infection with helper virus torecover the AAV—the required helper functions (i.e., adenovirus E1, E2a,VA, and E4 or herpesvirus ULS, ULB, UL52, and UL29, and herpesviruspolymerase) are also supplied, in trans, by the system. In these newersystems, the helper functions can be supplied by transient transfectionof the cells with constructs that encode the required helper functions,or the cells can be engineered to stably contain genes encoding thehelper functions, the expression of which can be controlled at thetranscriptional or posttranscriptional level. In yet another system, thetransgene flanked by ITRs and rep/cap genes are introduced into insectcells by infection with baculovirus-based vectors. For reviews on theseproduction systems, see generally, e.g., Zhang et al., 2009,“Adenovirus-adeno-associated virus hybrid for large-scale recombinantadeno-associated virus production,” Human Gene Therapy 20:922-929, thecontents of each of which is incorporated herein by reference in itsentirety. Methods of making and using these and other AAV productionsystems are also described in the following U.S. patents, the contentsof each of which is incorporated herein by reference in its entirety:U.S. Pat. Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213;6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898;7,229,823; and 7,439,065. See generally, e.g., Grieger & Samulski, 2005,“Adeno-associated virus as a gene therapy vector: Vector development,production and clinical applications,” Adv. Biochem. Engin/Biotechnol.99: 119-145; Buning et al., 2008, “Recent developments inadeno-associated virus vector technology,” J. Gene Med. 10:717-733; andthe references cited below, each of which is incorporated herein byreference in its entirety. The methods used to construct any embodimentof this invention are known to those with skill in nucleic acidmanipulation and include genetic engineering, recombinant engineering,and synthetic techniques. See, e.g., Green and Sambrook et al, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (2012). Similarly, methods of generating rAAV virions arewell known and the selection of a suitable method is not a limitation onthe present invention. See, e.g., K. Fisher et al, (1993) J. Virol.,70:520-532 and U.S. Pat. No. 5,478,745.

Optionally, the hLDLR genes described herein may be delivered via viralvectors other than rAAV. Such other viral vectors may include any virussuitable for gene therapy may be used, including but not limited toadenovirus; herpes virus; lentivirus; retrovirus; etc. Suitably, whereone of these other vectors is generated, it is produced as areplication-defective viral vector.

A “replication-defective virus” or “viral vector” refers to a syntheticor artificial viral particle in which an expression cassette containinga gene of interest is packaged in a viral capsid or envelope, where anyviral genomic sequences also packaged within the viral capsid orenvelope are replication-deficient; i.e., they cannot generate progenyvirions but retain the ability to infect target cells. In oneembodiment, the genome of the viral vector does not include genesencoding the enzymes required to replicate (the genome can be engineeredto be “gutless”—containing only the transgene of interest flanked by thesignals required for amplification and packaging of the artificialgenome), but these genes may be supplied during production. Therefore,it is deemed safe for use in gene therapy since replication andinfection by progeny virions cannot occur except in the presence of theviral enzyme required for replication.

The pharmaceutical compositions described herein are designed fordelivery to subjects in need thereof by any suitable route or acombination of different routes. Direct delivery to the liver(optionally via intravenous, via the hepatic artery, or by transplant),oral, inhalation, intranasal, intratracheal, intraarterial, intraocular,intravenous, intramuscular, subcutaneous, intradermal, and otherparental routes of administration. The viral vectors described hereinmay be delivered in a single composition or multiple compositions.Optionally, two or more different AAV may be delivered, or multipleviruses [see, e.g., WO 2011/126808 and WO 2013/049493]. In anotherembodiment, multiple viruses may contain different replication-defectiveviruses (e.g., AAV and adenovirus).

The replication-defective viruses can be formulated with aphysiologically acceptable carrier for use in gene transfer and genetherapy applications. In the case of AAV viral vectors, quantificationof the genome copies (“GC”) may be used as the measure of the dosecontained in the formulation. Any method known in the art can be used todetermine the genome copy (GC) number of the replication-defective viruscompositions of the invention. One method for performing AAV GC numbertitration is as follows: Purified AAV vector samples are first treatedwith DNase to eliminate un-encapsidated AAV genome DNA or contaminatingplasmid DNA from the production process. The DNase resistant particlesare then subjected to heat treatment to release the genome from thecapsid. The released genomes are then quantitated by real-time PCR usingprimer/probe sets targeting specific region of the viral genome (usuallypoly A signal).

Also, the replication-defective virus compositions can be formulated indosage units to contain an amount of replication-defective virus that isin the range of about 1.0×10⁹ GC to about 1.0×10¹⁵ GC (to treat anaverage subject of 70 kg in body weight), and preferably 1.0×10¹² GC to1.0×10¹⁴ GC for a human patient. In another embodiment, the dose is lessthan about 1.5×10¹¹ GC/kg. For example, the dose of AAV virus may beabout 1×10⁹ GC, about 5×10⁹ GC, about 1×10¹⁰ GC, about 5×10¹⁰ GC, orabout 1×10¹¹ GC. In another example, the variants may be delivered in anamount of about 0.001 mg to about 10 mg/kg.

The above-described recombinant vectors may be delivered to host cellsaccording to published methods. The rAAV, preferably suspended in aphysiologically compatible carrier, may be administered to a human ornon-human mammalian subject. Suitable carriers may be readily selectedby one of skill in the art in view of the indication for which thetransfer virus is directed. For example, one suitable carrier includessaline, which may be formulated with a variety of buffering solutions(e.g., phosphate buffered saline). Other exemplary carriers includesterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran,agar, pectin, peanut oil, sesame oil, and water. The selection of thecarrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and/or variants and carrier(s), other conventionalpharmaceutical ingredients, such as preservatives, or chemicalstabilizers. Suitable exemplary preservatives include chlorobutanol,potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, theparabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable chemical stabilizers include gelatin and albumin.

The viral vectors and other constructs described herein may be used inpreparing a medicament for delivering a LDLR variant to a subject inneed thereof, supplying LDLR variant having an increased half-life to asubject, and/or for treating elevated cholesterol levels, elevated highdensity lipoprotein (HDL), elevated triglycerides, familialhypercholesterolemia, atherosclerosis, coronary artery disease,cardiovascular disease, and/or another lipoprotein metabolic disorder.

A course of treatment may optionally involve repeat administration ofthe same viral vector (e.g., an AAV8 vector) or a different viral vector(e.g., an AAV8 and an AAVrh10). Still other combinations may be selectedusing the viral vectors described herein. Optionally, the compositiondescribed herein may be combined in a regimen involving other anti-lipiddrugs (e.g., statins, monoclonal antibodies, etc), or protein-basedtherapies (including, e.g., delivery of a composition containing one ormore LDLR variants as described herein).

It is to be noted that the term “a” or “an” refers to one or more. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively. While various embodiments in the specificationare presented using “comprising” language, under other circumstances, arelated embodiment is also intended to be interpreted and describedusing “consisting of” or “consisting essentially of” language.

As used herein, the term “about” means a variability of 10% from thereference given, unless otherwise specified.

The term “regulation” or variations thereof as used herein refers to theability of a composition to inhibit one or more components of abiological pathway.

A “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog,cat, horse, cow, pig, or non-human primate, such as a monkey,chimpanzee, baboon or gorilla.

As used herein, “disease”, “disorder” and “condition” are usedinterchangeably, to indicate an abnormal state in a subject.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art and by reference to published texts, whichprovide one skilled in the art with a general guide to many of the termsused in the present application.

The following examples are illustrative only and are not intended tolimit the present invention.

EXAMPLES Example 1—AAV Vectors Expressing LDLR Gain-of-Function VariantsDemonstrate Increased Efficacy in Mouse Models of FamilialHypercholesterolemia

A. Experimental Animals

All animal studies were approved by the institutional review board (IRB)at the University of Pennsylvania. LDLR^(−/−), APOBEC-1^(−/−) doubleknockout mice (DKO) and LDLR^(−/−), APOBEC-1^(−/−), human ApoB100transgenic (LAHB) were maintained at the University of Pennsylvania.These mice overexpress hPCSK9. The absence of endogenous mouse LDLRexpression in this animal model permits evaluation of hLDLR transgeneexpression without interference from mouse LDLR. Overexpression ofhPCSK9 is achieved by coadministering an AAV vector expressing hPCSK9(AAV9.TBG.hPCSK9), the preparation of which is described in Part C ofthis Example.

6-8 week old male mice were injected intravenously (tail vein) withvector diluted in phosphate buffered saline (PBS) in a total volume of100 μL. Serum was collected pre and post vector administration by retroorbital bleeds. At the end of the study all animals were sacrificed andthe livers harvested for analysis of vector genomes and transgeneexpression. Serum samples from animals were analyzed for totalcholesterol (Tc), LDL, HDL and total triglycerides (Tg) using a MIRAanalyzer (Roche). Non-HDL cholesterol was derived by subtracting the Tgfrom Tc. Livers from animals were harvested and homogenized using RIPAbuffer. 25 μg of total liver lysate was electrophoresed on a 4-12% PAGEgel and probes with a polyclonal anti-hLDLR antibody.

B. LDLR Variants

Amino acid residues (position numbers based on SEQ ID NO:1) targeted formutagenesis were as follows:

TABLE 1 Amino acid substitutions and affected LDLR-PCSK9 interactionAmino acid substitution Predicted LDLR-PCSK9 interaction N295D Preventhydrogen bonding with PCSK9 Asp-238 D299N Affects salt bridge with PCSK9Ser-153 H306G Affects salt bridge with PCSK9 Asp-374 V307D Preventshydrophobic interaction with PCSK9 Val-380 N309A Prevent hydrogenbonding with PCSK9 Thr-377 D310N Affects salt bridge with PCSK9 Arg-194L311T Prevent hydrogen bonding with PCSK9 Thr-377 L318D Hydrophobicinteraction with PCSK9 Cys-378 L318H Hydrophobic interaction with PCSK9Cys-378

In addition, amino acid substitutions, K809R and C818A, in theC-terminal cytoplasmic domain of LDLR that prevent IDOL mediateddegradation were selected.

C. Vector

The AAV8 vector expressing wild type hLDLR cDNA from a liver-specificthyroxine binding globulin (TBG) promoter has been previously describedand was obtained from the Vector Core at the University of Pennsylvania.Briefly, HEK293 cells were triple transfected using AAV cis- andtrans-plasmid along with the Ad helper plasmids. AAV particles werepurified from the culture supernatant and quantified using primers tothe bGH polyadenylation sequence. Vector preparations were analyzed forDNA structure by restriction digests and endotoxin contamination (<20EU/mL) before injection into animals. The wild type hLDLR cDNA was usedas a template for site directed mutagenesis to introduce amino acidsubstitutions using the Quickchange XL kit (Stratagene) as per themanufacturers' recommendations.

The sequences of plasmids used for production of AAV vectors asdescribed herein are provided in the appended sequence listing. Theplasmid constructs having the TBG promoter were used in the animal(mice) studies; those with the CB promoter were used for in vitroscreening.

The cDNA sequences encoding hPCSK9 and hIDOL were purchased (Origene,Md.), cloned, and vectored to express from an AAV9 vector behind a TBGpromoter and a bovine growth hormone (bGH) polyadenylation signal. AnAAV9 vector expressing human alpha1-antitrypsin (A1AT) also expressedfrom a TBG promoter was used as a control in studies that required anirrelevant transgene.

TABLE 2 Vectors AAV8.hLDLR AAV8.hLDLR-N295D AAV8.hLDLR-D299NAAV8.hLDLR-H306G AAV8.hLDLR-V307D AAV8.hLDLR-N309A AAV8.hLDLR-D310NAAV8.hLDLR-L311T AAV8.hLDLR-L318D AAV8.hLDLR-L318HAAV8.hLDLR-K809R\C818A AAV8.hLDLR-L318D\K809R\C818A AAV9.hPCSK9AAV9.hlDOL

D. In Vitro LDLR Assay

HEK 293 cells growing in 6 well plates were transfected overnight withplasmids expressing hLDLR along with hPCSK9 or hIDOL. All cDNAs werecloned behind a cytomegalovirus promoter (CMV) to obtain expression inHEK293 cells. Control cells were transfected with hLDLR plus a plasmidexpressing an irrelevant transgene (A1AT). In studies where the dose ofone vector was titrated lower an irrelevant plasmid was added to ensurethat the total amount of plasmid did not vary from one experimental wellto another. The following day cells were pulsed with BODIPY-LDL(Invitrogen) at a concentration of 4 μg/mL. Cells were removed after 2hr and evaluated for fluorescent LDL uptake using a flow cytometer(FC500, Beckman Coulter).

E. Immunoblotting and Enzyme Linked Immune Assays

50 μg of total cell lysates prepared from cells or mouse liversexpressing human LDLR were electrophoresed on a 4-12% gradient precastmini gel (Invitrogen) before transferring to PVDF membrane (Invitrogen).An anti-hLDLR goat polyclonal antibody (Invitrogen) was used to probethe membrane ( 1/1000 dilution) followed by a secondary anti-goatantibody conjugated to alkaline phosphatase (Invitrogen). Human PCSK9expression levels in mouse serum were analyzed using an ELISA kit (R&D)as per the manufacturers' instructions.

F. Statistical Analysis

All experiments were analyzed using one-way Analysis of Variance modelswith pair-wise group differences in mean cholesterol level assessedusing Tukey's post-hoc tests. However, for experiments evaluating theeffect of PCSK9 in C57BL/6 mice, a linear mixed effects model was usedto assess group differences in cholesterol level while taking intoaccount correlation between repeated measurements on the same mouse.Similarly, analysis of PCSK9 on AAV transduced hLDLR relied on Analysisof Covariance modeling, with post-cholesterol level regressed onpre-cholesterol level and group. Statistical significance was taken atthe 0.05 level for all experiments.

Results

G. Amino Acid Substitutions in hLDLR Confer PCSK9 Resistance

Nine LDLR variants with potentially decreased binding to PCSK9 (N295D,D299N, H306G, V307D, N309A, D310N, L311T, L318D and L318H see Table 1,position numbers based on numbering of SEQ ID NO:1) were initiallyscreened in HEK293 cells using an in vitro assay for uptake offluorescently labeled-LDL (BODPIY-LDL™), in the presence or absence ofhPCSK9.

Studies in HEK293 cells that have low levels of endogenous expression ofhLDLR and hPCSK9 were performed. As a source of exogenous hPCSK9, cellswere co-transfected with a plasmid expressing hPCSK9 along with thehLDLR constructs. Mock transfected cells expressed low levels of LDLRbased on immunoblotting which failed to detect LDLR protein (data notshown); moreover, mock transfected cells failed to demonstrate uptake ofBODIPY™-LDL (FIG. 1A). In contrast, transient transfection of wild typehLDLR into HEK293 cells led to internalization of BODIPY™-LDL in 30% ofcells which was reduced to 18% when co-transfected with hPCSK9 (FIG.1A). Among the mutant constructs co-expressed with hPCSK9, only theD299N and L311T amino acid substitutions failed to afford any protectionto PCSK9 mediated degradation in that BODIPY™-LDL uptake was reduced toa similar extent as wild type LDLR. All other amino acid substitutionsafforded varying degrees of protection from PCSK9, although someconstructs were less efficient in BODIPY™-LDL uptake in the absence ofPCSK9 when compared to wild-type hLDLR. As an example, although theL318D and L318H substitutions were both resistant to hPCSK9 degradation,only L318D showed normal BODIPY™-LDL uptake in the absence of PCSK9(FIG. 1B). In contrast, the L318H substitution led to reduced receptoractivity and BODIPY™-LDL uptake was lower when compared to wild typehLDLR in the absence of hPCSK9 (30% vs 6%; hLDLR vs hLDLR-L318H). Forthis reason the hLDLR-L318D vector was selected for further in vivoevaluation in mice.

H. Overexpression of hPCSK9 in Mice Downregulates AAV Expressed hLDLR

Evaluating the activity of wildtype and L318D forms of hLDLR in mice wascomplicated because of potential diminished interactions between theexogenous hLDLR protein and the endogenous mouse PCSK9 protein. A fullyhumanized mouse model with the hoFH phenotype (lacking LDLR and APOBEC-1by virtue of germ line interruption) and overexpressing hPCSK9[following i.v. injection of an AAV9 vector expressing hPCSK9 via theliver specific promoter TBG (AAV9.hPCSK9)] was created (the LDLR−/−,ApoBec −/− double knock-out (DKO) mice described in Part A of thisExample. Expression of AAV9.hPCSK9 vector was first evaluated in C57BL/6mice who received increasing doses of hPCSK9. At high dose vector (i.e.,5×10¹⁰ GC) serum non-HDL cholesterol increased approximately 2.5-fold(p=0.0015), indicating some level of interaction between hPCSK9 andmLDLR. See, FIG. 1C.

Prior to evaluating the effects of hPCSK9 on transgene derived hLDLR,the hoFH DKO mice were injected with AAV8.hLDLR alone. In these animals,baseline non-HDL levels on a chow diet were 417±23 mg/dl; whichdecreased by day 7 following administration of 5×10¹⁰ GC of AAV8.hLDLR.Non-HDL levels stabilized and were only 37±7 mg/dl by day 30 which was9% of baseline levels (p=0.037, FIG. 2A). Next, the performance of thisvector in DKO mice expressing hPCSK9 were evaluated by co-administering(i.v.) an equal dose (5×10¹⁰ GC) of AAV9.hPCSK9 along with AAV8.hLDLR.Following vector administration, serum levels of hPCSK9 rose steadilyand reached peak levels (7500±3000 ng/mL) by day 30 (FIG. 2B).Concomitantly, non-HDL levels in mice co-transduced with hPCSK9 weresignificantly higher (p=0.0008) when compared to animals that onlyreceived hLDLR (FIG. 2A). AAV8.hLDLR reduced non-HDL 10-fold in theabsence of hPCSK9; however, this reduction was only 2.5-fold in thepresence of hPCSK9. Immunoblotting of total liver lysates confirmed thatco-transduction with PCSK9 resulted in reduced hLDLR protein in theliver (FIG. 2C); whereas, levels of hLDLR messenger RNA remainedunchanged between the experimental groups (data not shown). Thesefindings are consistent with the reported mode of action of PCSK9 tobind and sequester LDLR in an intracellular compartment that increasesreceptor degradation [Wang, et al, J Lipid Res, 2012; 53: 1932-1943]. Noreduction in hLDLR expression was observed in animals co-transduced withan AAV9 vector expressing an irrelevant transgene.

I. THE LDLR-L318D Amino Acid Substitution Confers Resistance to HumanPCSK9 Mediated Degradation

A similar strategy was used to evaluate the activity of hLDLR-L318D inDKO mice overexpressing hPCSK9 and compared the results to micetransduced with wild type hLDLR. As expected, transduction with hLDLRresulted in a dramatic lowering of serum cholesterol by day 30 (10% ofbaseline); while, co-transduction with hPCSK9 resulted in reduced hLDLRactivity with non-HDL cholesterol levels only 23% of baseline (p<0.0001,FIG. 3A). In contrast, the L318D substitution apparently preventedreceptor degradation in that differences in non-HDL levels betweenanimals that received hLDLR-L318D or hLDLR-L318D along with hPCSK9 wasnot statistically significant (10% vs 14%; p=0.1337). Moreover,immunoblotting of livers collected at the end of the study (Day 30)revealed that hLDLR protein levels were significantly decreased only inanimals that received wild-type hLDLR along with hPCSK9 but not in thosethat received hLDLR alone (FIG. 3B). However, liver levels ofhLDLR-L318D were unaffected by co-expression with hPCSK9 and the same asobserved with wild type hLDLR in the absence of hPCSK9 (FIG. 3B). Toconfirm that the observed differences did not arise from changes in mRNAexpression, hLDLR transcripts were analyzed in livers using aquantitative PCR assay. These studies indicated only a modest decreasein wild type hLDLR treated mice that was substantially less than thedecrease in hLDLR protein (FIG. 3B).

J. hLDLR-K809R\C818A Escapes hIDOL Regulation

LDLR expression is also subject to regulation by IDOL; an E3 ubiquitinligase transcriptionally unregulated by liver X receptors (LXRs)following an increase in intracellular concentrations of oxysterols.Activated IDOL interacts with the cytoplasmic tail region of LDLRleading to receptor degradation [Zhang L, et al, Arterioscler ThrombVasc Biol. 2012; 32:2541-2546]. An AAV8 vector expressing hLDLRcontaining the K809R and C818A amino acid substitutions(AAV8.hLDLR-K808R\C818A) was constructed. This construct was firstevaluated in HEK293 cells in the presence or absence of hIDOL; as asource of human IDOL, plasmids expressing hIDOL were co-expressed withhLDLR. As expected, transfection of wild type hLDLR resulted in LDLuptake in 28% of cells; however, co-transfection of hIDOL along withhLDLR dramatically reduced LDL positive cells to only 2% (FIG. 4A). TheK808R\C818A amino acid substitutions did not impact receptor activityand the LDLR-K809R\C818A construct was as efficient as wild type hLDLRin internalizing LDL, in the absence of IDOL (LDLR vs LDLR-K809R\C818A,28% vs 22%). However, differences between the two constructs did appearwhen co-transfected with hIDOL. The hLDLR-K809R\C818A construct was moreresistant to the effects of hIDOL resulting in roughly 14% of cellstaking up fluorescent LDL as opposed to 2% with wild type LDLR.Immunoblotting of whole cell lysates further confirmed that the observeddifferences in LDL uptake correlated with reduced levels of hLDLRprotein, and not hLDLR-K809R\C818A, in the presence of hIDOL (FIG. 4A).

Next the activity of the hLDLR-K809R\C818A construct in DKO miceoverexpressing human IDOL was evaluated. A phenotype of miceoverexpressing human IDOL in liver was created by administering an AAV9vector expressing human IDOL under control of a liver specific promoter.In pilot studies the efficacy of human IDOL in regulating endogenousLDLR expression was evaluated in mice by administering (i.v.) 5×10¹⁰ GCof AAV9.hIDOL to FH mice heterozygous for LDLR expression (heFH). Thisstrain of mice (LAHB mice) is deficient in APOBEC-1, heterozygous formouse LDLR′ and transgenic for human ApoB100 which leads to higher serumcholesterol. Following administration of AAV9.hIDOL, non-HDL levelsincreased by day 7 and reached stable levels by day 30 (p<0.0001, FIG.4B). These results confirmed that AAV expressed hIDOL was active inmouse livers and can cause the loss of endogenous mLDLR. Next, theeffect of hIDOL overexpression on vector encoded hLDLR was expressed inDKO mice. In pilot studies only low dose hLDLR vector administrationswere significantly impacted by human IDOL; hence, mice wereco-administered 3×10⁹ GC of AAV8.hLDLR and 5×10¹⁰ GC of AAV9.hIDOL. Atthis low dose, hLDLR and hLDLR-K809\C818A vectors were functionallysimilar (p=0.9) and induced a modest reduction (20% of baseline) inserum cholesterol in the absence of hIDOL (FIG. 4C). However,co-administration of hIDOL ablated wild type hLDLR activity and nocorrection was seen in non-HDL cholesterol levels which remained atpre-treatment baseline levels (p=0.0248, FIG. 4C). In contrast, non-HDLcholesterol levels in mice that received hLDLR-K809R\C818A in thepresence or absence of hIDOL were similar (p>0.05) demonstrating the invivo resistance of the modified constructs to hIDOL (FIG. 4C).

Example 2—hLDLR-L318D\K809R\C818A Avoids Regulation by Both PCSK9 andIDOL

The L318D, K809R and C818A amino acid substitutions were cloned into asingle vector to create a construct that would be resistant toregulation by both pathways. The vector was administered to DKO mice ata low dose (3×10⁹ GC), when evaluating the IDOL escape mutations; or ata higher dose (5×10¹⁰ GC), when evaluating the PCSK9 escape mutation.When administered at a low dose, hLDLR-L318D\K809R\C818A was comparableto wild type hLDLR (p>0.05) in that only a modest decrease in serumcholesterol was realized following either vector administration (FIG.5A). However when administered in the presence of hIDOL, only the mutantvector showed any resistance to hIDOL in that serum cholesterol levelsremained significantly lower than that seen in wild type hLDLR plushIDOL (p=0.0002). Immunoblotting of liver samples confirmed that themutant vector was more resistant to hIDOL mediated degradation (FIG.5A). In the parallel study where vectors were administered at a higherdose along with hPCSK9, the variant protein performed significantlybetter in reducing serum cholesterol than the control wild type LDLR inmice overexpressing hPCSK9 (p=0.0007, FIG. 5B). Immunoblot analysis oflivers demonstrated a nearly complete absence of wild type hLDLR in thepresence of hPCSK9; in contrast, the mutant vector was protected andless degraded by hPCSK9.

Example 3—Comparison of hLDLR Variants in a Mouse Model of FamilialHypercholesterolemia

The panel of hLDLR carrying single amino acid substitutions that wereexpected to avoid PCSK9 regulation were screened by administering toLDLR−/−, APOBEC−/− double knockout mice (DKOs). Animals were injectedintravenously (i.v. tail vein) with 3×10¹⁰ GC of AAV8.TBG.hLDLR or oneof the hLDLR variants that was expected to avoid hPCSK9 regulation.Reduction in serum levels of non-HDL cholesterol was used as a surrogatefor comparing receptor activity from the different constructs. Serum wascollected from animals by retro-orbital bleeds before and 30 days aftervector administration and cholesterol levels analyzed using a MIRAanalyzer (Roche). Non-HDL cholesterol levels were determined bysubtracting the HDL component from total cholesterol. FIG. 6 showspercent decline in non-HDL levels over baseline in animals followingvector administration.

This study was repeated under the same conditions, with the exceptionthat vector administered at a higher dose, i.e., 5×10¹⁰ GC ofAAV8.TBGF.hLDLR for each of variants (L318D, N295D, H306G, V307D, N309A,D310N, L311T, L318H). Administration of 5×10¹⁰ GC of wild type hLDLR byitself led to a 90% decrease in baseline non-HDL cholesterol levels(FIG. 8 ). With the exception of D299N, all other hLDLR variants alsoachieved similar reduction in non-HDL cholesterol. As expected,coadministration of hPCSK9 significantly reduced the efficacy of hLDLRvector. hPCSK9 overexpression had only a minimal effect on variants,L318D, N295D, H306G, V307D and N309A. Furthermore, immunoblotting of day30 livers confirmed that with the exception of the H306G, these variantswere significantly protected from degradation (not shown).

Example 4—High Dose AAV.hLDLR Administration Circumvents In Vivo IDOLInhibition

LDLR−/−, APOBEC−/− double knockout mice (DKOs) were injected withAAV8.TBG.hLDLR or AAV8.TBG.K809R\C818A at a dose of 5×10¹⁰ GC. Inaddition, some groups of mice were co-administered with an equal dose ofan AAV9 vector expressing human IDOL (AAV9.TBG.hIDOL) to evaluate hLDLRactivity in the presence of hIDOL. Non-HDL cholesterol levels wereanalyzed before and 30 days after vector administration. The percentnon-HDL cholesterol at day 30 compared to baseline following vectoradministration is shown in FIG. 7 .

All publications cited in this specification are incorporated herein byreference, as are US Provisional Patent Application Nos. 62/022,627,filed Jul. 9, 2014 and 61/984,620, filed Apr. 25, 2014. Similarly, theSEQ ID NOs which are referenced herein and which appear in the appendedSequence Listing are incorporated by reference. While the invention hasbeen described with reference to particular embodiments, it will beappreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the appended claims.

1. A recombinant vector having an expression cassette comprising amodified human low density lipoprotein receptor (hLDLR) gene, whereinsaid hLDLR gene encodes a modified hLDLR that reduces cholesterolfollowing expression, and wherein said modified hLDLR comprises: (a) oneor more amino acid substitutions that interfere with the wild-type hLDLRIDOL pathway; and/or (b) one or more amino acid substitutions which areresistant to degradation of hLDLR by interfering with the PCSK9 pathway.2. The recombinant vector of claim 1, wherein the modified hLDLRcomprises (a) an amino acid substitution at one or more of N295, H306,V307, N309, D310, and/or L318, based on the numbering of SEQ ID NO:1. 3.The recombinant vector of claim 2, wherein the hLDLR comprises one ormore amino acid substitutions selected from: N295D, H306D, V307D, N309A,D310N, L318H, and/or L318D, based on the numbering of SEQ ID NO:1. 4.The recombinant vector of claim 1, wherein the modified hLDLR comprises(b) an amino acid substitution of at least one of L769R, K809R and/orC818A, based on the numbering of SEQ ID NO:1, optionally in combinationwith an amino acid substitution of (a).
 5. The recombinant vector ofclaim 1, wherein the vector is a recombinant adeno-associated virus(rAAV) vector.
 6. The recombinant vector of claim 1, wherein the vectoris a rAAV comprising a capsid selected from AAV8, rh64R1, AAV9, or rh10.7. The recombinant vector of claim 1, wherein the expression cassettecomprises a promoter which specifically directs expression of themodified hLDLr in liver cells.
 8. A rAAV vector having an expressioncassette comprising a modified hLDLR gene, wherein said hLDLR geneencodes a modified hLDLR comprising an L318D amino acid substitution,based on the numbering of SEQ ID NO:1.
 9. The rAAV vector of claim 8,wherein the modified hLDLR further comprises a K809R and/or C818A aminoacid substitution, based on the numbering of SEQ ID NO:1.
 10. The rAAVvector of claim 8, wherein the vector comprises a capsid selected fromAAV8, rh64R1, AAV9, or rh10.
 11. The rAAV vector of claim 8, wherein theexpression cassette comprises a promoter which specifically directsexpression of the modified hLDLr in liver cells.
 12. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and arecombinant vector of claim
 1. 13. A method for reducing circulatingcholesterol levels by administering to a subject in need thereof arecombinant vector of claim 1, wherein said expression cassette furthercomprises regulatory control sequences which direct expression ofmodified hLDLR in the subject.
 14. A method for reducing circulatingcholesterol levels by administering to a subject in need thereof a rAAVvector of claim 8, wherein said expression cassette further comprisesregulatory control sequences which direct expression of modified hLDLRin the subject.
 15. A synthetic or recombinant hLDLR comprising: (a) anamino acid substitution at one or more of N295, H306, V307, N309, D310,L318, and/or L318, based on the numbering of SEQ ID NO:1; or (b) anamino acid substitution of any of (a) in combination with an amino acidsubstitution of at least one of K769R, K809R and/or C818A, based on thenumbering of SEQ ID NO:1.
 16. The synthetic or recombinant hLDLR ofclaim 15, wherein the one or more amino acid substitutions of (a) areselected from: N295D, H306D, V307D, N309A, D310N, L318H, and/or L318D,based on the numbering of SEQ ID NO:1.
 17. A pharmaceutical compositioncomprising a synthetic or recombinant hLDLR protein of claim 15 and apharmaceutically acceptable carrier.